Arabidopsis 2010: The Function of Subtilase Genes in Plant Development

摘要本研究旨在明确拟南芥枯草杆菌酶基因的功能。 枯草杆菌酶基因编码枯草杆菌蛋白酶样丝氨酸蛋白酶，这种蛋白质在植物生长和发育中起重要作用。 在哺乳动物系统中，一些枯草杆菌酶被称为“激素转化酶”，因为它们切割其他蛋白质，释放生物活性肽，所述生物活性肽充当激素和/或在细胞间信号传导中起作用。 一些植物枯草杆菌酶的性质表明它们可能在植物发育和生理中发挥类似的作用。 通过与拟南芥枯草杆菌酶联盟（TASC）的欧洲合作伙伴的合作，将研究拟南芥中近60个枯草杆菌酶基因的功能。 枯草杆菌酶基因家族中的所有基因的列表（及其亚家族鉴定）可见于http://csbdb.mpimp-golm.mpg.de/csbdb/dbcawp/psdb/main/mgenes.html。 本研究将集中在10个枯草杆菌酶基因的作用，在组织培养中的芽再生过程中高度调节。 这些枯草杆菌酶基因中的三个似乎影响芽发育，因为这些基因的表达受决定芽再生效率的主要数量性状位点（QTL）控制。 拟南芥和一些重要的经济作物的芽再生效率各不相同，这可能会阻碍基因工程技术的成功应用。 另外7个枯草杆菌酶基因在芽或根再生过程中差异调节。蛋白质组学方法（使用蛋白质表达谱研究大量蛋白质）将用于了解枯草杆菌酶的功能。通过定量分析T-DNA插入突变体和诱导表达枯草杆菌酶基因的转基因体的二维差异凝胶电泳（DIGE）图谱，将检查这些基因在体内的功能。 枯草杆菌酶靶位点将通过分析重组酶对常见底物蛋白的有限消化产物来确定。 通过设计和测试与常见底物切割位点相关的合成肽的水解，确定最佳枯草杆菌酶靶位点。研究结果将通过http://csbdb.mpimp-golm.mpg.de/csbdb/dbcawp/psdb.html与公众和科学界其他成员分享。 本网站的信息由Altmann实验室编辑，并将每月更新。 这项研究对2010年项目的目标具有重要意义，因为它有助于我们了解一组影响植物生长和发育的重要基因。 这项研究作出了独特的贡献，因为它使用蛋白质组学的新工具来解决基因功能。更广泛的影响这个项目具有更广泛的影响，因为它有助于了解蛋白质水解和肽信号在植物发育中的作用。 对基因组学中的生物活性植物肽知之甚少，因为肽通常嵌入其他蛋白质中，并且直到被蛋白酶（如枯草杆菌酶）切割才释放。 该项目还具有更广泛的影响，因为它为爱荷华州州立大学和得梅因地区社区学院的本科生提供实验室研究培训。 一些本科学员也将有机会在夏季在与TASC相关的合作伙伴的国际实验室进行研究。为了鼓励ISU少数民族本科生参加这个项目，我们将每年秋季在罗伊·J·卡弗联合实验室为农业，自然资源和相关科学（MANRRS）少数民族的地方分会举办一次会议。

Abstract This research will determine the function of subtilase genes in Arabidopsis. Subtilase genes encode subtilisin-like serine proteases, proteins that play important roles in plant growth and development. In mammalian systems, some subtilases are called "hormone convertases" because they cut other proteins releasing bioactive peptides that serve as hormones and/or function in cell-to-cell signaling. The properties of some plant subtilases suggest they may play similar roles in plant development and physiology. The function of nearly 60 subtilase genes in Arabidopsis will be investigated through collaboration with European partners in The Arabidopsis Subtilase Consortium (TASC). A listing of all the genes in the subtilase gene family (and their subfamily identification) is found at http://csbdb.mpimp-golm.mpg.de/csbdb/dbcawp/psdb/main/mgenes.html. This research will focus on the role of ten subtilase genes that are highly regulated during shoot regeneration in tissue culture. Three of these subtilase genes appear to influence shoot development in that the expression of these genes is controlled by a major quantitative trait locus (QTL) that determines shoot regeneration efficiency. Shoot regeneration efficiency varies in Arabidopsis and in some important economic crops where it can impede the successful application of genetic engineering technologies. Another seven subtilase genes are differentially regulated during shoot or root regeneration. A proteomics approach (the study of a large number of proteins using protein expression profiling) will be used to understand the function of the subtilases. The function of these genes in vivo will be examined by quantitatively analyzing 2D difference gel electrophoresis (DIGE) profiles of T-DNA insertion mutants and transgenics with induced expression of subtilase genes. Subtilase target sites will be determined by analyzing the products from the limited digestion of common substrate proteins by the recombinant enzyme. Optimal subtilase target sites will be determined by designing and testing the hydrolysis of synthetic peptides related to the cleavage sites for common substrates.Research results will shared with the public and other members of the scientific community through http://csbdb.mpimp-golm.mpg.de/csbdb/dbcawp/psdb.html. Information at this site is edited by the Altmann lab and will be updated on a monthly basis. This research is significant to the goals of the 2010 project in that it contributes to our understanding of a group of important genes that influence plant growth and development. This research makes a unique contribution in that it uses new tools in proteomics to address gene function.Broader impactThis project has broader impact because it contributes knowledge to the role of proteolysis and peptide signaling in plant development. Little is known about bioactive plant peptides from genomics because peptides are often embedded in other proteins and are not released until cleaved by proteases, such as subtilases. The project also has broader impact because it provides lab research training for Iowa State University and Des Moines Area Community College undergraduates. Some of the undergraduate trainees will also have the opportunity to conduct research during the summer in international labs of partners associated with TASC. To encourage ISU minority undergrads to participate in this project, we will host a meeting each fall in the Roy J. Carver Co-Laboratory for the local chapter of Minorities in Agriculture, Natural Resources and Related Sciences (MANRRS).